Alpha-agarase and process for producing the same

ABSTRACT

A polypeptide having an α-agarase activity, a gene encoding the polypeptide, a method for producing the polypeptide by genetic engineering and a method for producing an agarooligosaccharide using the polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of PCTinternational application No. PCT/JP00/00966 which has an internationalfiling date of Feb. 21, 2000 which designated the United States, theentire contents of which are incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an α-agarase and a method for producingthe same. Specifically, the present invention relates to an α-agarase,which is useful for producing agarooligosaccharides with low degrees ofpolymerization having various physiological activities from agarose, anda method for producing the α-agarase as well as use of the enzyme. Thepresent invention also relates to a polypeptide having an α-agaraseactivity and a gene encoding said polypeptide. Specifically, the presentinvention relates to an amino acid sequence of an α-agarase, which isuseful for producing agarooligosaccharides with low degrees ofpolymerization having various physiological activities from agarose, anda nucleotide sequence encoding the amino acid sequence. Furthermore, thepresent invention relates to a method for producing a polypeptide havingan α-agarase activity by genetic engineering. In addition, the presentinvention relates to a method for producing an agarooligosaccharideusing a polypeptide having an α-agarase activity.

2. Description of Related Art

Agarose is the principal constituent of agar. Agarose is apolysaccharide that has a structure in which D-galactose and3,6-anhydro-L-galactose are alternately linked together through α-1,3bonds and β-1,4 bonds. One must degrade agarose into smaller moleculesin order to produce oligosacchaides from agar. For this purpose, methodsin which agarose is chemically degraded and methods in which agarose isenzymatically digested are known. In a chemical degradation method,agarose can be hydrolyzed using an acid. In this case, α-1,3 bonds aremainly cleaved. Two enzymes, β-agarase which cleaves β-1,4 bonds inagarose and α-agarase which cleaves α-1,3 bonds in agarose, are known todigest agarose.

Oligosaccharides obtained by cleaving agarose at β-1,4 bonds are calledas neoagarooligosaccharides. Neoagarooligosaccharides have D-galactoseat their reducing ends and their degrees of polymerization are expressedby even numbers. On the other hand, oligosaccharides obtained bycleaving agarose at α-1,3 bonds are called as agarooligosaccharides.Agarooligosaccharides have 3,6-anhydro-L-galactose at their reducingends and their degrees of polymerization are expressed by even numbers.Recently, it was shown that agarooligosaccharides which have3,6-anhydro-L-galactose at their reducing ends have physiologicalactivities such as an apoptosis-inducing activity, a carcinostaticactivity, various antioxidant activities, an immunoregulatory activity,an antiallergic activity, an anti-inflammatory activity and an activityof inhibiting α-glycosidase (WO99/24447, Japanese Patent Application No.11-11646). Based on the physiological activities, pharmaceuticalcompositions and functional foods or drinks containing theagarooligosaccharides as their active ingredients can be provided.

It is difficult to control the size of produced oligosaccharides in amethod in which agarose is chemically degraded. In particular, it isquite difficult to selectively produce smaller oligosaccharides with lowdegrees of polymerization (e.g., T. Tokunaga et al., Bioscience &Industry, 49:734 (1991)). If β-agarase is used, onlyneoagarooligosaccharides which do not have the above-mentionedphysiological activities can be obtained because this enzyme cleavesonly β-1,4 bonds.

It is expected that agarooligosaccharides having physiologicalactivities are produced by using α-agarase which has an activity ofcleaving α-1,3 bonds. Known α-agarases include enzymes produced by amarine Gram-negative bacterial strain GJ1B (Carbohydrate Research,66:207-212 (1978); this strain is indicated as Alteromonas agarlyticusstrain GJ1B in European Journal of Biochemistry, 214:599-607 (1993)) anda bacterium of genus Vibrio (JP-A 7-322878; strain JT0107-L4). However,it is impossible to produce agarobiose which has notable physiologicalactivities by using the α-agarase derived from Alteromonas agarlyticusstrain GJ1B because the enzyme cannot digest hexasaccharides or shorteroligosaccharides. Furthermore, the α-agarase derived from a bacterium ofgenus Vibrio cannot be used for the production of agarooligosaccharidesusing agarose as a raw material because this enzyme exhibits itsactivity only on hexasaccharides and shorter oligosaccharides and doesnot act on agarose at all.

As described above, prior art has problems regarding the production ofsmaller agarooligosaccharides such as agarobiose and agarotetraose whichhave 3,6-anhydro-L-galactose at their reducing ends and have variousphysiological activities.

The main object of the present invention is to provide a polypeptidehaving an α-agarase activity which can be used for efficient productionof smaller agarooligosaccharides, an amino acid sequence of thepolypeptide, a gene encoding the polypeptide, a method for producing thepolypeptide and a method for producing the smalleragarooligosaccharides.

SUMMARY OF THE INVENTION

In view of the problems as described above, the present inventors havestudied intensively and conducted search in order to obtain an enzymethat cleaves α-1,3 bonds in agarose and generates agarooligosaccharideshaving notable physiological activities. As a result, the presentinventors have successfully found two microbial strains that produceenzymes having properties suitable for this purpose. The enzymesproduced by these microorganisms were isolated and their physical andchemical as well as enzymatic properties were elucidated. Furthermore,the present inventors have successfully isolated genes for the enzymes,and found a method for readily producing polypeptides having α-agaraseactivities by means of genetic engineering using the genes, therebycompleting the present invention.

The present invention is outlined as follows. The first aspect of thepresent invention relates to a novel α-agarase having the followingphysical and chemical properties:

(1) action: hydrolyzing an α-1,3 bond between 3,6-anhydro-L-galactoseand D-galactose;

(2) substrate specificity: acting on agarose, agarohexaose andagarooligosaccharides longer than agarohexaose but not on agarotetraose;

(3) optimal temperature: exhibiting its enzymatic activity at atemperature of 55° C. or below; and (4) heat stability: retaining 20% ormore of its activity after treatment at 48° C. for 30 seconds.

Such α-agarases are exemplified by an enzyme that contains an amino acidsequence consisting of 749 residues from amino acid number 177 to aminoacid number 925 in the amino acid sequence of SEQ ID NO:14, or an aminoacid sequence in which one or more amino acids are substituted, deleted,added and/or inserted in said amino acid sequence consisting of 749residues, or an enzyme that contains an amino acid sequence consistingof 767 residues from amino acid number 184 to amino acid number 950 inthe amino acid sequence of SEQ ID NO:15, or an amino acid sequence inwhich one or more amino acids are substituted, deleted, added and/orinserted in said amino acid sequence consisting of 767 residues.

The second aspect of the present invention relates to a gene encoding apolypeptide having an α-agarase activity, which encodes the α-agarase ofthe first aspect. Such genes are exemplified by a gene that contains anucleotide sequence consisting of 2247 bases from base number 529 tobase number 2775 in the nucleotide sequence of SEQ ID NO:12, or anucleotide sequence in which one or more bases are substituted, deleted,added and/or inserted in said nucleotide sequence consisting of 2247bases, or a gene that contains a nucleotide sequence consisting of 2301bases from base number 550 to base number 2850 in the nucleotidesequence of SEQ ID NO:13, or a nucleotide sequence in which one or morebases are substituted, deleted, added and/or inserted in said nucleotidesequence consisting of 2301 bases.

The third aspect of the present invention relates to a gene that ishybridizable to the gene of the second aspect under stringent conditionsand encodes the α-agarase of the first aspect.

The fourth aspect of the present invention relates to a recombinant DNAmolecule that contains the gene of the second or third aspect.

The fifth aspect of the present invention relates to a transformantharboring the recombinant DNA molecule of the fourth aspect.

The sixth aspect of the present invention relates to a method forproducing a polypeptide having an α-agarase activity, comprisingculturing a microorganism capable of producing an α-agarase (e.g., amicroorganism belonging to a genus to which a microorganism TKR1-7AGα(FERM BP-6990) or a microorganism TKR4-3AGα (FERM BP-6991) belongs) andcollecting the α-agarase of the first aspect from the culture. Both ofthe microorganisms TKR1-7AGα and TKR4-3AGα were deposited under BudapestTreaty on Jan. 26, 1999 (the date of the original deposit) at theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan underaccession numbers FERM BP-6990 and FERM BP-6991, respectively.

The seventh aspect of the present invention relates to a method forproducing a polypeptide having an α-agarase activity, comprisingculturing the transformant of the fifth aspect and collecting theα-agarase of the first aspect from the culture.

The eighth aspect of the present invention relates to a method forproducing an agarooligosaccharide, comprising digesting agarose usingthe α-agarase of the first aspect and collecting an agalooligosaccharidefrom the resulting digest.

The ninth aspect of the present invention relates to a novel α-agarase.Such α-agarases are exemplified by an enzyme containing an amino acidsequence consisting of 591 residues from amino acid number 335 to aminoacid number 925 in the amino acid sequence of SEQ ID NO: 14, or an aminoacid sequence in which one or more amino acids are substituted, deleted,added and/or inserted in said amino acid sequence consisting of 591residues, or an enzyme containing an amino acid sequence consisting of586 residues from amino acid number 365 to amino acid number 950 in theamino acid sequence of SEQ ID NO: 15, or an amino acid sequence in whichone or more amino acids are substituted, deleted, added and/or insertedin said amino acid sequence consisting of 586 residues.

The tenth aspect of the present invention relates to a gene encoding apolypeptide having an α-agarase activity, which encodes the α-agarase ofthe ninth aspect. Such genes are exemplified by a gene containing anucleotide sequence consisting of 1773 bases from base number 1003 tobase number 2775 in the nucleotide sequence of SEQ ID NO: 12, or anucleotide sequence in which one or more bases are substituted, deleted,added and/or inserted in said nucleotide sequence consisting of 1773bases, or a gene containing a nucleotide sequence consisting of 1758bases from base number 1093 to base number 2850 in the nucleotidesequence of SEQ ID NO: 13, or a nucleotide sequence in which one or morebases are substituted, deleted, added and/or inserted in said nucleotidesequence consisting of 1758 bases.

The eleventh aspect of the present invention relates to a gene that ishybridizable to the gene of the tenth aspect under stringent conditionsand encodes the α-agarase of the ninth aspect.

The twelfth aspect of the present invention relates to a recombinant DNAmolecule that contains the gene of the tenth or the eleventh aspect.

The thirteenth aspect of the present invention relates to a transformantharboring the recombinant DNA molecule of the twelfth aspect.

The fourteenth aspect of the present invention relates to a method forproducing a polypeptide having an α-agarase activity, comprisingculturing the transformant of the thirteenth aspect and collecting theα-agarase of the ninth aspect from the culture.

The fifteenth aspect of the present invention relates to a method forproducing an agarooligosaccharide, comprising digesting agarose usingthe α-agarase of the ninth aspect and collecting an agarooligosaccharidefrom the resulting digest.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates digestion of agarohexaose by the α-agarase of thepresent invention.

FIG. 2 illustrates digestion of agarohexaose by the α-agarase of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, an oligosaccharide refers to a saccharide composed of 2or more and 10 or less monosaccharides. An agarooligosaccharide refersto an oligosaccharide that has a structure in which D-galactose and3,6-anhydro-L-galactose are alternately linked together through α-1,3bonds and β-1,4 bonds and has 3,6-anhydro-L-galactose at its reducingend. A polysaccharide refers to a saccharide other than monosaccharidesand oligosaccharides.

The α-agarase of the present invention is an enzyme that hydrolyzes anα-1,3 bond between 3,6-anhydro-L-galactose and D-galactose. It can acton both polysaccharides such as agarose and oligosaccharides such asagarohexaose. The enzymes of the present invention are not specificallylimited as long as they have such properties. Examples thereof includean α-agarase produced by a marine microorganism TKR1-7AGα (FERM BP-6990)or TKR4-3AGα (FERM BP-6991).

Agarase 1-7 produced by the microorganism TKR1-7AGα and Agarase 4-3produced by the microorganism TKR4-3AGα are enzymes that hydrolyze α-1,3bonds between 3,6-anhydro-L-galactose and D-galactose in polysaccharidesand oligosaccharides. These enzymes act on agarose, agarohexaose andagarooligosaccharides longer than agarohexaose as well asneoagarohexaose and neoagarooligosaccharides longer thanneoagarohexaose.

A method for measuring the activities of the above-mentioned twoenzymes, and physical and chemical as well as enzymatic properties ofthe enzymes are described below.

(1) Method for Measuring Enzymatic Activity

The activity of the α-agarase of the present invention is measured byconducting an enzymatic reaction using agarose as a substrate and thenquantifying the resulting agarotetraose. Specifically, the method formeasuring an enzymatic activity used herein for measuring the activityof a purified enzyme preparation and an enzyme in the course ofpurification is as follows.

A solution containing agarose (Takara Shuzo, Code: 5003) at aconcentration of 0.2% in 10 mM tris-hydrochloride (pH 7.0), 10 mMcalcium chloride and 10 mM sodium chloride is prepared. 180 μl of thissolution as a substrate is mixed with 20 μl of an enzyme solution. Themixture is reacted at 42° C. for 30 to 120 minutes, preferably 60minutes, and then heated at 60° C. for 1 minute to stop the reaction. 30μl of the reaction mixture is subjected to a TSKgel α-2500 column (innerdiameter: 7.8 mm; length: 300 mm; Tosoh, Code: 18339). A peak is elutedat retention time of about 26 minutes using 70% acetonitlrile solutionas an eluent at a flow rate of 0.8 ml/minute. Agarotetraose produced asa result of the enzymatic reaction in the peak is quantified. One unit(1 U) is defined as the amount of the enzyme that produces 1 micromoleof agarotetraose in 10 minutes.

(2) Optimal pH

An enzyme was allowed to act on agarose as a substrate in a reactionmixture prepared using an acetate buffer (pH 4.5), a malate buffer (pH5.5), an acetate buffer (pH 6.0, 6.5) or a tris-hydrochloride buffer (pH7.0, 7.5, 8.8). As a result, it was demonstrated that Agarase 1-7 andAgarase 4-3 exhibit their activities of digesting agarose under neutralto weakly acidic conditions and under weakly alkaline to weakly acidicconditions, respectively.

(3) Optimal Temperature

The enzyme of the present invention exhibits its enzymatic activity at atemperature of 55° C. or below. It exhibits a high activity at atemperature ranging from 30 to 48° C., and exhibits the maximal activityat about 37 to 42° C.

(4) Heat Stability

Remaining activities of enzyme preparations after treatment at 48° C.,50° C. or 60° C. for 30 seconds were measured. As a result, Agarase 1-7exhibited 25% of its activity after treatment at 48° C., and Agarase 4-3exhibited 22% of its activity after treatment at 50° C.

(5) Molecular Weight

The molecular weight of Agarase 1-7 was estimated to be about 95,000 asdetermined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a10-20% polyacrylamide gradient gel.

The molecular weight of Agarase 4-3 was determined by equilibriumdensity-gradient centrifugation using glycerol density gradient andSDS-PAGE. As a result, the molecular weight of Agarase 4-3 was estimatedto be about 85,000.

(6) Amino Acid Sequencing by Edman Degradation Method

The N-terminal amino acid sequences of Agarase 1-7 and Agarase 4-3 asdetermined by Edman degradation method wereAsp-Thr-Leu-Ser-Val-Glu-Ala-Glu-Met-Phe andGly-Asp-Ile-Val-Ile-Glu-Leu-Glu-Asp-Phe-Asp-Ala-Thr-Gly-Thr-Thr-Gly-Arg-Val-Ala,respectively. The N-terminal amino acid sequences of Agarase 1-7 andAgarase 4-3 are shown in SEQ ID NOS: 1 and 2, respectively.

As described below, both of a gene encoding Agarase 1-7 and a geneencoding Agarase 4-3 were isolated. Also, amino acid sequences encodedby these genes were determined. Amino acid sequences encoded by theAgarase 1-7 gene and the Agarase 4-3 gene are shown in SEQ ID NOS: 14and 15, respectively. Both of a polypeptide consisting of 749 aminoacids from amino acid number 177 to amino acid number 925 in the aminoacid sequence of SEQ ID NO:14 and a polypeptide consisting of 767 aminoacids from amino acid number 184 to amino acid number 950 in the aminoacid sequence of SEQ ID NO:15 exhibit the activities of the α-agarase ofthe present invention.

Furthermore, each of the following polypeptides also exhibits anactivity of the α-agarase of the present invention: a polypeptideconsisting of 725 amino acids from amino acid number 201 to amino acidnumber 925 in the amino acid sequence of SEQ ID NO: 14; a polypeptideconsisting of 591 amino acids from amino acid number 335 to amino acidnumber 925 in the amino acid sequence of SEQ ID NO: 14; a polypeptideconsisting of 700 amino acids from amino acid number 251 to amino acidnumber 950 in the amino acid sequence of SEQ ID NO: 15; and apolypeptide consisting of 586 amino acids from amino acid number 365 toamino acid number 950 in the amino acid sequence of SEQ ID NO: 15.

As described below, the α-agarase of the present invention can beproduced by genetic engineering using the gene for the α-agarase. IfEscherichia coli, Bacillus subtilis or the like, which is widely usedfor production of proteins by genetic engineering, is used as a host, itis considered that the possibility of arising the following problemsbecomes higher as the molecular weight of the foreign protein to beexpressed becomes higher:

(1) when the host is changed to another, the expressed protein may forman inclusion body and be insolubilized;

(2) the expression level may be reduced due to the preference of thehost for amino acid codons;

(3) if the molecular weight of the protein to be expressed is higherthan that of a protein normally contained in the host, the protein maybecome unstable, the expression level may be low, and the protein may bedegraded in some cases; and

(4) the protein may be inactivated being subjected to othermodifications.

Thus, it is important to determine the minimal polypeptide portionrequired for exhibiting the activity of interest of the protein to beproduced.

It is considered that the minimal portions for exhibiting the α-agaraseactivities for the agarases of the present invention, Agarase 1-7 andAgarase 4-3, are a polypeptide consisting of 591 amino acids from aminoacid number 335 to amino acid number 925 in the amino acid sequence ofSEQ ID NO: 14, and a polypeptide consisting of 586 amino acids fromamino acid number 365 to amino acid number 950 in the amino acidsequence of SEQ ID NO: 15, respectively.

The α-agarase of the present invention can be purified from a cultureobtained by culturing a microorganism TKR1-7AGα (FERM BP-6990) or amicroorganism TKR4-3AGα (FERM BP-6991). TKR1-7AGα and TKR4-3AGα wereisolated from seawater as bacteria that assimilate agar. They have thefollowing microbiological properties.

Microbiological properties:

(1) Morphology

100 ml of artificial seawater (product name: Jamarine S; JamarineLaboratory) was prepared. 0.3 g of peptone (DIFCO, Code: 00123-17-3) and0.02 g of yeast extract (DIFCO, Code: 0127-17-9) were added thereto. ThepH was then adjusted to 8.0 with 3M sodium carbonate. The resultingmixture was transferred into a 500-ml Erlenmeyer flask. 0.1 g of agar(Nacalai Tesque, Code: 010-28) was added thereto. After sterilization inan autoclave, one of the above-mentioned microorganisms was inoculatedinto the mixture, and cultured at 25° C. at 120 rpm overnight. The cellsof both TKR1-7AGα and TKR4-3AGα grown in the cultures were bacillary andmotile.

(2) Growth

100 ml of artificial seawater (product name: Jamarine S; JamarineLaboratory) was prepared. 0.3 g of peptone and 0.02 g of yeast extractwere added thereto. The pH was then adjusted to 8.0 with 3M sodiumcarbonate. 1.5 g of agar (Nacalai Tesque, Code: 010-28) was addedthereto. The mixture was autoclaved to prepare plates. When theabove-mentioned microorganisms were inoculated onto the plates, it wasdemonstrated that:

(a) they grow well at 23 to 30° C.; and

(b) the agar gel is liquefied as the cells grow.

100 ml of artificial seawater (product name: Jamarine S; JamarineLaboratory) was prepared. 0.3 g of peptone and 0.02 g of yeast extractwere added thereto. The pH was then adjusted to a varying value with 3Msodium carbonate. The resulting mixture was transferred into a 500-mlErlenmeyer flask. 0.1 g of agar was added thereto. After sterilizationin an autoclave, one of the above-mentioned microorganisms wasinoculated into the mixture. Then, it was demonstrated that:

(c) they grow well at pH 7.0 to 8.5.

TKR1-7AGα and TKR4-3AGα were deposited under Budapest Treaty on Jan. 26,1999 (the date of the original deposit) at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry under accessionnumbers FERM BP-6990 and FERM BP-6991, respectively.

Information on the taxonomic position of each of the above-mentionedmicroorganisms can be obtained by analyzing the nucleotide sequence ofthe gene encoding 16S ribosomal RNA of which the nucleotide sequence isknown to be specific for each microbial strain. Specifically,chromosomal DNAs are extracted from the microbial strains TKR1-7AGα andTKR4-3AGα. PCRs are carried out using primers that can be used toamplify a region of the gene or a portion thereof. Nucleotide sequencesof the amplified DNA fragments are determined. Homology search utilizingGenBank database is conducted against the determined sequences. Then,microorganisms that have similar nucleotide sequences in the region,i.e., taxonomically close microorganisms, can be known.

DNA fragments derived from 16S ribosomal RNA genes in the microorganismsTKR1-7AGα and TKR4-3AGα were amplified according to a method asdescribed in Bulletin of Japanese Society of Microbial Ecology, 10:31-42(1995). Primers 27f and 1492r described in the literature were used forthe PCRs (nucleotide sequences of the primers 27f and 1492r are shown inSEQ ID NOS:16 and 17). The nucleotide sequences of the resultingamplified DNA fragments were analyzed. As a result, the followingmicroorganisms each having a homology of about 95% in theabove-mentioned region with one of the microorganisms producing theα-agarase of the present invention were found:

TKR1-7AGα: Colwellia like bacterium;

TKR4-3AGα: Marine psychrophile IC079.

A medium containing a nitrogen source, an inorganic substance and thelike that can be utilized by each of the microorganisms as well as agar,agarose or the like as a carbon source can be used as a medium forculturing the microorganism. Commercially available agar and agarose canbe used. Examples of nitrogen sources include meat extract, yeastextract, casein hydrolysate, tryptone, peptone and the like. Yeastextract and peptone are preferably used. Such a nitrogen source can alsobe used as a carbon source in addition to agar or agarose. Furthermore,sodium chloride, iron citrate, magnesium chloride, sodium sulfate,calcium chloride, potassium chloride, sodium carbonate, sodiumbicarbonate, potassium bromide, strontium chloride, sodium borate,sodium silicate, sodium fluoride, ammonium nitrate, disodiumhydrogenphosphate and the like can be used in combination as salts.

In particular, a medium prepared by adding peptone, yeast extract andagar or agarose to a medium consisting of artificial seawater Jamarine Scan be preferably used. It is preferable to add agar or agarose at aconcentration of 0.1 to 2.0%. A solid or liquid medium can be preparedby appropriately changing the concentration of agar or agarose. Liquidculture using a concentration of 0.1 to 0.3% is preferable for thepurpose of production of enzyme, whereas solid culture using aconcentration of 1.2 to 2.0% is preferable for the purpose ofpreservation of cells. If low melting point agarose is used for liquidculture, it can be used at a concentration of 0.1 to 1.0%.

Although culture conditions vary more or less depending on thecomposition of the medium, the cultivation temperature is 23 to 30° C.,preferably 25° C., the pH of the medium is 7.0 to 8.5, preferably 7.2 to8.2, and the cultivation time is 12 to 48 hours, preferably 24 to 36hours.

The α-agarase of the present invention produced during cultivation asdescribed above is secreted outside the cells. Then, the cells areremoved after cultivation by means of centrifugation, filtration or thelike to obtain a culture supernatant.

The resulting culture supernatant can be concentrated using vacuumconcentration or ultrafiltration to prepare a liquid enzyme.Alternatively, the culture supernatant can be converted to a powderyenzyme by lyophilization, spray-drying or the like to prepare a crudeenzyme preparation. The α-agarase of the present invention can bepartially purified by a conventional purification method such as saltingout with ammonium sulfate or solvent precipitation. Furthermore, apurified enzyme preparation which results in a single band uponelectrophoresis can be obtained using known purification procedures suchas column chromatographies (e.g., anion-exchange column and gelfiltration column) in combination.

Agarooligosaccharides such as agarobiose, agarotetraose and agarohexaosecan be produced by reacting the thus-obtained culture or the α-agaraseof the present invention in a varying degree of purification with apolysaccharide contained in red algae such as agar or agarose as asubstrate.

Agarose is a polysaccharide that has a structure in which D-galactoseand 3,6-anhydro-L-galactose are alternately linked together throughα-1,3 bonds and β-1,4 bonds. β-agarase is an enzyme that hydrolyzesβ-1,4 bonds in agarose. Oligosaccharides having D-galactose at theirreducing ends generated by the action of this enzyme are called asneoagarooligosaccharides, which do not exhibit physiological activitiessuch as those observed for agarooligosaccharides. If an α-agarase thatcleaves α-1,3 bonds in agarose is used, agarooligosaccharides having3,6-anhydro-L-galactose at their reducing ends can be produced. Twoenzymes derived from Alteromonas agarlyticus strain GJ1B and a bacteriumof genus Vibrio (strain JT0107-L4) are known as α-agarases. However, theα-agarase produced by Alteromonas agarlyticus strain GJ1B cannot act onagarohexaose or shorter oligosaccharides, whereas the α-agarase derivedfrom the bacterium of genus Vibrio cannot digest agarose. Thus, it wasimpossible to efficiently produce agarobiose or agarotetraose fromagarose a raw material using a known α-agarase.

The α-agarase of the present invention is an enzyme that acts onagarose, agarohexaose and agarooligosaccharides longer than agarohexaoseas seen from the above-mentioned physical and chemical properties. Thus,it acts on agarose to produce agarooligosaccharides. Furthermore, it cancleave the single α-1,3 bond in agarohexaose generated as a result ofthe action. In other words, by allowing the α-agarase of the presentinvention to act on agarose, it is possible to obtain agarobiose andagarotetraose, a disaccharide and a tetrasaccharide which have beenscarcely produced according to conventional methods, in largequantities.

Substrate specificities of the known α-agarases and the α-agarase of thepresent invention are shown in Table 1. In the table, the marks + and −represent the ability and inability of the enzyme to digest thesubstrate, respectively. TABLE 1 Alteromonas Bacterium of agarlyticusgenus Vibrio Agarase 1-7 Substrate GJ1B (JT0107-L4) Agarase 4-3Agarose + − + Agarohexaose − + + Agarotetraose − + −

The α-agarase of the present invention acts on agarohexaose to generateagarobiose and agarotetraose.

The agarooligosaccharides produced using the α-agarase of the presentinvention contain agarobiose and agarotetraose. Theagarooligosaccharides may contain agarohexaose or agarooligosaccharideslonger than agarohexaose as long as the existence does not interferewith the purpose of use. Agar, agarose, or oligosaccharides derived fromagar or agarose may be used as a raw material for the production ofagarobiose, agarotetraose and agarohexaose using the α-agarase of thepresent invention. The conditions used for the action of the α-agaraseare not specifically limited as long as the enzyme exhibits its activityunder the conditions used. For example, it is preferable to allowAgarase 1-7 to act under neutral to weakly acidic conditions, to allowAgarase 4-3 to act under weakly alkaline to weakly acidic conditions,and to allow both of the enzymes to act at 37 to 42° C. The compositionof the reaction mixture is not specifically limited as long as it issuitable for the action of the enzyme.

As described above, the oligosaccharides produced using the α-agarase ofthe present invention are mainly hexasaccharides or shorteroligosaccharides with low degrees of polymerization. However, it ispossible to optionally produce oligosaccharides with different degreesof polymerization by appropriately selecting the reaction conditions orthe like. It is also possible to obtain agarobiose, agarotetraose andagarohexaose independently by separating and purifying the thus-obtainedoligosaccharides.

The α-agarase gene of the present invention is a gene that encodes apolypeptide having the above-mentioned α-agarase activity. Thus, itrefers to a nucleic acid that contains a nucleotide sequence encoding anamino acid sequence for a polypeptide having an α-agarase activity.Examples of the α-agarase genes of the present invention include a genethat contains a nucleotide sequence encoding Agarase 1-7 derived fromthe microorganism TKR1-7AGα (FERM BP-6990) and a gene that contains anucleotide sequence encoding Agarase 4-3 derived from the microorganismTKR4-3AGα (FERM BP-6991). Genes encoding Agarase 1-7 and Agarase 4-3 areexemplified by a gene having a nucleotide sequence encoding apolypeptide consisting of 749 amino acid residues from amino acid number177 to amino acid number 925 in SEQ ID NO:14, and a gene having anucleotide sequence encoding a polypeptide consisting of 767 amino acidresidues from amino acid number 184 to amino acid number 950 in SEQ IDNO:15.

In addition, the α-agarase genes of the present invention areexemplified by a gene encoding a polypeptide consisting of 725 aminoacid residues from amino acid number 201 to amino acid number 925 in SEQID NO: 14; a gene having a nucleotide sequence encoding a polypeptideconsisting of 591 amino acid residues from amino acid number 335 toamino acid number 925 in SEQ ID NO: 14; a gene encoding a polypeptideconsisting of 700 amino acid residues from amino acid number 251 toamino acid number 950 in SEQ ID NO: 15; and a gene having a nucleotidesequence encoding a polypeptide consisting of 586 amino acid residuesfrom amino acid number 365 to amino acid number 950 in SEQ ID NO: 15.

The α-agarase genes of the present invention also include a nucleic acidthat contains a nucleotide sequence encoding a polypeptide that has anamino acid sequence in which one or more amino acids are substituted,deleted, added and/or inserted in the above-mentioned amino acidsequence and has an α-agarase activity.

Also, the genes of the present invention include a gene having anucleotide sequence consisting of 2247 bases from base number 529 tobase number 2775 in SEQ ID NO:12, and a gene having a nucleotidesequence consisting of 2301 bases from base number 550 to base number2850 in SEQ ID NO:13.

The present invention also encompasses a gene having a nucleotidesequence consisting of 2175 bases from base number 601 to base number2775 in SEQ ID NO: 12, a gene having a nucleotide sequence consisting of1773 bases from base number 1003 to base number 2775 in SEQ ID NO: 12; agene having a nucleotide sequence consisting of 2100 bases from basenumber 751 to base number 2850 in SEQ ID NO: 13; and a gene having anucleotide sequence consisting of 1758 bases from base number 1093 tobase number 2850 in SEQ ID NO: 13.

Furthermore, the genes of the present invention also include a nucleicacid containing a nucleotide sequence that has a nucleotide sequence inwhich one or more bases are substituted, deleted, added and/or insertedin the above-mentioned nucleotide sequence and encodes a polypeptidehaving an α-agarase activity.

Furthermore, the genes of the resent invention include a nucleic acidcontaining a nucleotide sequence that hybridizes to the above-mentionedgene under stringent conditions and encodes a polypeptide having anα-agarase activity. Hybridization can be carried out according to amethod as described in, for example, T. Maniatis et al. (eds.),Molecular Cloning: A Laboratory Manual 2nd ed., 1989, Cold Spring HarborLaboratory. The stringent conditions are exemplified by incubation witha probe at 65° C. overnight in a solution containing 6×SSC (1×SSC: 0.15M NaCl, 0.015 sodium citrate, pH 7.0), 0.5% SDS, 5× Denhardt's and 100mg/ml herring sperm DNA.

For example, the α-agarase gene of the present invention can be clonedas follows.

A genomic DNA is prepared from a microorganism producing an α-agarase.The genomic DNA can be prepared according to an appropriate knownmethod. For example, it can be prepared using known procedures such aslysozyme treatment, protease treatment, RNase treatment, phenoltreatment and ethanol precipitation in combination. The thus-obtainedgenomic DNA is degraded by appropriate known means such as sonication ordigestion with a restriction enzyme. The resulting DNA fragments areincorporated into a vector (e.g., a plasmid vector) according to aconventional method to construct recombinant DNA molecules. Therecombinant DNA molecules are then introduced into an appropriate host(e.g., Escherichia coli) to obtain transformants. Procedures forconstruction of recombinant DNA molecules, transformation and the likecan be appropriately selected depending on the vector and the host to beused from conventional methods, for example, those described inMolecular Cloning: A Laboratory Manual 2nd ed. Thus, a genomic librarythat contains a transformant harboring an α-agarase gene is obtained.

Next, the genomic library is screened to select the transformantharboring the α-agarase gene. Examples of screening methods are asfollows.

(1) Screening Using Expression of α-Agarase Activity as Index

A genomic library is grown on agar plates. A transformant harboring anα-agarase gene expresses a polypeptide having an α-agarase activity. Thepolypeptide lyses agar gel by the action of its α-agarase activity.Accordingly, a colony or a plaque that lyses agar gel in an agar plateis selected.

(2) Screening Using Antibody

A crude, partially purified or purified enzyme preparation of anα-agarase is prepared as described above. An anti-α-agarase antibody isprepared using the preparation as an antigen according to a conventionalmethod.

A genomic library is grown on plates. Grown colonies or plaques aretransferred onto nylon or nitrocellulose filters. Expressed recombinantproteins are transferred onto the filters along with the colonies orplaques. The recombinant proteins on the filters are reacted with theanti-α-agarase antibody to identify a clone reactive with the antibody.

The clone reactive with the antibody can be identified according to aknown method, for example, by reacting a peroxidase-conjugated secondaryantibody with the filters which have been reacted with theanti-α-agarase antibody, incubating the filter with a chromogenicsubstrate and then detecting developed color.

If an expression vector that results in high expression of a gene in aDNA incorporated in the vector is used for the construction of thegenomic library to be used in the method (1) or (2) as described above,a transformant harboring the gene of interest can be readily selected.

(3) Screening by Hybridization using DNA Probe

A genomic library is grown on plates. Grown colonies or plaques aretransferred onto nylon or nitrocellulose filters. DNAs are immobilizedonto the filters by denaturation. The DNAs on the filters are hybridizedto a labeled probe according to a conventional method to identify aclone that hybridizes to the probe.

Probes used for this screening include an oligonucleotide prepared basedon information on the amino acid sequence of the α-agarase as describedabove, an oligonucleotide prepared based on information on another aminoacid sequence, and a PCR fragment amplified using primers prepared basedon information on such an amino acid sequence. Examples of labels usedfor the probes include, but are not limited to, a radioisotopic label, afluorescent label, a digoxigenin label and a biotin label.

A genomic library enriched for transformants harboring an α-agarase geneprepared as follows may be used as a genomic library to be used for thescreening.

A genomic DNA is prepared from a microorganism producing an α-agarase,digested with an appropriate restriction enzyme, separated by agarosegel electrophoresis and then blotted onto a nylon or nitrocellulosefilter according to a conventional method. The DNAs on the filter arehybridized to the above-mentioned labeled probe according to aconventional method to detect a DNA fragment that hybridizes to theprobe. DNA fragments corresponding to the signal are extracted andpurified from the agarose gel.

The thus-obtained DNA fragments are incorporated into a vector (e.g., aplasmid vector) according to a conventional method to constructrecombinant DNA molecules. The recombinant DNA molecules are thenintroduced into an appropriate host (e.g., Escherichia coli) to obtaintransformants. A transformation method suitable for the vector to beused can be selected from conventional methods, for example, thosedescribed in Molecular Cloning: A Laboratory Manual 2nd ed. Thus, agenomic library enriched for transformants harboring an α-agarase geneis obtained.

A screening can be carried out more efficiently by using such a genomiclibrary.

(4) In Vitro Cloning Using PCR

The gene of interest is cloned by screening transformants in the methods(1) to (3) as described above. By using a PCR, cloning can be carriedout in vitro without utilizing transformants.

A genomic DNA is prepared from a microorganism producing an α-agarase.The genomic DNA is degraded by appropriate known means such assonication or digestion with a restriction enzyme. Linkers are ligatedto the thus-obtained DNA fragments according to a conventional method.

A PCR is carried out using an oligonucleotide prepared based oninformation on an amino acid sequence of an α-agarase and anoligonucleotide that hybridizes to the linker as primers as well as thegenomic library as a template. The resulting amplification product isincorporated into a vector (e.g., a plasmid vector) according to aconventional method.

The nucleotide sequence of the α-agarase gene in the transformantharboring the α-agarase gene obtained as described above in (1) to (3)or the recombinant DNA molecule containing the α-agarase gene obtainedas described above in (4) can be determined according to a known method.If the clone does not encode the entire α-agarase polypeptide, theentire open reading frame for the α-agarase is revealed by repeating aprocedure comprising preparing a new probe based on the determinednucleotide sequence and screening a genomic library using the probe toobtain a new clone. A clone that contains the entire open reading frameencoding the α-agarase can be made based on the thus-obtainedinformation, for example.

A polypeptide having an α-agarase activity can be produced in largequantities by genetic engineering by connecting the gene encoding theα-agarase obtained as described above with an appropriate expressionvector.

A method for obtaining a gene for Agarase 1-7 is described below inbrief.

Cells obtained by culturing TKR1-7AGα are lysed using lysozyme, and thensubjected to removal of proteins, ethanol precipitation and the like toobtain a genomic DNA. The genomic DNA is partially digested with arestriction enzyme BamHI. The resulting DNA fragments are inserted intoa plasmid vector (ampicillin resistant) to construct a plasmid library.The plasmid library is used to transform Escherichia coli. Transformantsare grown on LB agar medium containing 1.5% (w/v) agar and 50 μg/mlampicillin and cultured at 37° C. for 5 days. After cultivation,colonies for which degradation of surrounding agar is observed areisolated, inoculated into LB medium containing ampicillin and cultured.An α-agarase activity is determined for a crude extract prepared fromthe cells. A plasmid DNA was extracted according to a conventionalmethod from a transformant for which an α-agarase activity was observed.The length of a DNA inserted in the plasmid DNA was determined to beabout 8 kb. This recombinant plasmid is designated as pAA1. Escherichiacoli transformed with the plasmid is designated and indicated asEscherichia coli JM109/pAA1, and deposited under Budapest Treaty on May26, 1999 (the date of the original deposit) at the National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan under accession number FERMBP-6992.

Analysis of the nucleotide sequence of the DNA fragment inserted in theplasmid pAA1 revealed that it contains an open reading frame (ORF) thatencodes a polypeptide consisting of 925 amino acids as a region encodingan α-agarase. The nucleotide sequence of the open reading frame and theamino acid sequence encoded by the open reading frame are shown in SEQID NOS: 12 and 14, respectively. Thus, the amino acid sequence of SEQ IDNO: 14 is an example of the agarase of the present invention. Comparisonof this amino acid sequence with the previously determined N-terminalamino acid sequence of Agarase 1-7, P1, shows that Agarase 1-7 is apolypeptide consisting of an amino acid sequence from amino acid number28 to amino acid number 925 in the amino acid sequence of SEQ ID NO:14,and that the amino acid sequence is encoded by a nucleotide sequencefrom base number 82 to base number 2778 (including the terminationcodon) in the nucleotide sequence of SEQ ID NO:12. The amino acidsequence consisting of the amino acids 1 to 27 in the amino acidsequence of the SEQ ID NO: 14 is considered to be a signal sequence.Furthermore, a polypeptide having an amino acid sequence consisting of749 residues from amino acid number 177 to amino acid number 925 in theamino acid sequence of SEQ ID NO:14 also has an activity of theα-agarase of the present invention.

A method for obtaining a gene for α-Agarase 4-3 is described below inbrief. Mixed primers 3 and 4 were prepared based on the N-terminal aminoacid sequence of Agarase 4-3, P2, represented by SEQ ID NO: 2. Thesequences of the mixed primers 3 and 4 are shown in SEQ ID NOS: 6 and 7,respectively.

A chromosomal DNA prepared from TKR4-3AGα as described above forα-Agarase 1-7 was completely digested with a restriction enzyme BamHI.BamHI linkers were ligated to the termini of the digested DNA. A PCR wascarried out using the resulting DNA as a template as well as the mixedprimer 3 and a primer C1 attached to LA PCR in vitro cloning kit (TakaraShuzo).

Next, a PCR was carried out using the thus-obtained product of theprimary PCR as a template as well as the mixed primer 4 and a primer C2attached to LA PCR in vitro cloning kit (Takara Shuzo). As a result, anamplification product of about 2 kb was observed. This DNA fragment wasdesignated as 4-3N.

The nucleotide sequences of the terminal regions of the amplifiedfragment were analyzed. Primers 6 and 7 of which the nucleotidesequences are shown in SEQ ID NOS: 8 and 9, respectively, were preparedbased on a nucleotide sequence corresponding to a region that encodesthe N-terminal portion of the agarase. Similarly, primers 8 and 9 ofwhich the nucleotide sequences are shown in SEQ ID NOS: 10 and 11,respectively, were prepared based on a nucleotide sequence correspondingto the C-terminal portion of the agarase.

When a PCR was carried out as described above for 4-3N except thatprimers 6 and 7 were used in place of the mixed primers 3 and 4, a DNAfragment of about 1.0 kb, 4-3UN, was obtained. Similarly, when a PCR wascarried out using primers 8 and 9, a DNA fragment of about 2.0 kb, 4-3C,was obtained. Analysis of the nucleotide sequences of the fragments4-3UN and 4-3C, while taking the previously determined nucleotidesequence of 4-3N in consideration, revealed an open reading frame thatencodes a polypeptide consisting of 951 amino acids. The nucleotidesequence of the open reading frame and the amino acid sequence encodedby the nucleotide sequence of the open reading frame are shown in SEQ IDNOS: 13 and 15, respectively. In 4-3UN, a nucleotide sequence for theamino acid sequence P2, an upstream nucleotide sequence encoding 183amino acids, and a further upstream SD-like sequence were found.

Thus, the amino acid sequence of SEQ ID NO: 15 is an example of theagarase of the present invention. Comparison of this amino acid sequencewith the previously determined N-terminal amino acid sequence of Agarase4-3 shows that Agarase 4-3 is a polypeptide consisting of an amino acidsequence from amino acid number 184 to amino acid number 951 in theamino acid sequence of SEQ ID NO:15, and that the amino acid sequence isencoded by a nucleotide sequence from base number 550 to base number2856 (including the termination codon) in the nucleotide sequence of SEQID NO:13.

The amino acid sequences of Agarase 1-7 and Agarase 4-3 obtained asdescribed above as well as the nucleotide sequences of the genesencoding these enzymes have no homology with the amino acid sequences ofβ-agarases, which are known agarose-digesting enzymes that cleaveagarose in a different manner from the agarase of the present invention,and the nucleotide sequences of the genes encoding the enzymes. Thus,these sequences are considered to be absolutely novel.

A recombinant DNA molecule can be constructed by connecting a geneencoding the α-agarase of the present invention (e.g., the gene encodingAgarase 1-7 or Agarase 4-3) to an appropriate vector. Furthermore, atransformant can be made by introducing the recombinant DNA moleculeinto an appropriate host. The α-agarase of the present invention isproduced in a culture obtained by culturing the transformant. Thus, itis possible to produce the α-agarase of the present invention (e.g.,Agarase 1-7 or Agarase 4-3) in large quantities using the transformant.

A mutated α-agarase can be produced by introducing a mutation into agene encoding an α-agarase according to a known method. Examples of themethods for introducing a mutation that can be used include, but are notlimited to, the oligonucleotide double amber method (Hashimoto-Gotoh, T.et al., Gene, 152:271-275 (1995)), the gapped duplex method (Kramer, W.et al., Nucl. Acids Res., 12:9441 (1984); Kramer, W. et al., Methods inEnzymology, 154:350 (1987)) and the Kunkel method (Kunkel, T. A., Proc.Natl. Acad. Sci. USA, 82:488 (1985); Kunkel, T. A., Methods inEnzymology, 154:367 (1987)).

The α-agarase genes having nucleotide sequences of SEQ ID NOS: 12 and 13were deleted from the 5′-termini. The polypeptides encoded by theresulting deleted genes were expressed. The α-agarase activities werethen determined for the polypeptides. As a result, it was demonstratedthat each of the following genes encodes a polypeptide having anactivity of the α-agarase of the present invention: a gene having anucleotide sequence consisting of 2247 bases from base number 529 tobase number 2775 in SEQ ID NO: 12; a gene having a nucleotide sequenceconsisting of 2175 bases from base number 601 to base number 2775 in SEQID NO: 12; a gene having a nucleotide sequence consisting of 1773 basesfrom base number 1003 to base number 2775 in SEQ ID NO: 12; a genehaving a nucleotide sequence consisting of 2301 bases from base number550 to base number 2850 in SEQ ID NO: 13; a gene having a nucleotidesequence consisting of 2100 bases from base number 751 to base number2850 in SEQ ID NO: 13; and a gene having a nucleotide sequenceconsisting of 1758 bases from base number 1093 to base number 2850 inSEQ ID NO: 13.

The α-agarase of interest can be secreted outside a transformant byexpressing a gene that encodes a polypeptide in which a signal sequenceis added at the N-terminus of the α-agarase to be expressed. The signalsequence is not limited to specific one, and exemplified by the signalsequence for α-Agarase 1-7 represented by amino acid numbers 1 to 27 inSEQ ID NO: 14. This signal sequence is encoded by a nucleotide sequencefrom base number 1 to base number 81 in SEQ ID NO: 12.

Examples of vectors that can be used for constructing the recombinantDNA molecules include, but are not limited to, plasmid vectors, phagevectors and virus vectors. An appropriate vector may be selecteddepending on the purpose for which the recombinant DNA is used. In casewhere a recombinant DNA molecule is constructed in order to produce anα-agarase, a vector that contains a promoter and/or other regions forexpression control is preferable. Examples of such plasmid vectorsinclude, but are not limited to, pKF19k, pT7BlueT and pET16b. Hosts thatcan be used for making transformants include, but are not limited to,microorganisms such as bacteria, yeasts and filamentous fungi, as wellas cultured cells of mammals, fishes, insects and the like. Arecombinant DNA molecule constructed using a vector suitable for thehost is used for making a transformant.

A method for producing Agarase 1-7 by genetic engineering is describedbelow in brief.

A DNA fragment containing a nucleotide sequence that encodes apolypeptide having, for example, an amino acid sequence starting fromamino acid number 28 or 177 in SEQ ID NO: 14 is amplified by a PCR usingthe plasmid pAA1 which encodes the α-Agarase 1-7 gene as a template toconstruct a plasmid in which the amplified fragment is inserted into anappropriate plasmid vector (e.g., pKF19k (Takara Shuzo), pT7BlueT(Takara Shuzo) or pET16b (Takara Shuzo)). Escherichia coli (e.g.,Escherichia coli JM109 or BL21(DE3)pLysS) transformed with such aplasmid is cultured in an appropriate liquid medium. Induction iscarried out using IPTG or the like, if necessary. The polypeptideencoded by the DNA fragment inserted in the plasmid is expressed. Theα-agarase activity expressed by such a transformant in a unit volume ofculture is usually higher than that observed for a culture of TKR1-7AGα.

For Agarase 4-3, a transformant expressing the α-agarase can be made bygenetic engineering as described above for Agarase 1-7 after amplifyinga DNA fragment containing a nucleotide sequence that encodes, forexample, a polypeptide having an amino acid sequence starting from aminoacid number 184 in SEQ ID NO: 15 by a PCR using a chromosomal DNA fromthe microorganism TKR4-3a as a template. The resulting transformantexpresses an α-agarase activity, and the activity in a unit volume ofculture is usually higher than that observed for a culture of TKR4-3AGα.

The α-agarase of the present invention produced by genetic engineeringas described above can be partially purified by a conventionalpurification method such as salting out with ammonium sulfate or solventprecipitation. Furthermore, a purified enzyme preparation which resultsin a single band upon electrophoresis can be obtained using knownpurification procedures such as column chromatographies (e.g.,anion-exchange column and gel filtration column) in combination.

Agarooligosaccharides such as agarobiose, agarotetraose and agarohexaosecan be produced by reacting the thus-obtained recombinant α-agarase ofthe present invention in a varying degree of purification with apolysaccharide contained in red algae such as agar or agarose as asubstrate.

The following Examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

EXAMPLE 1

Production of α-Agarase from TKR1-7AGα

Upon purification of the α-agarase of the present invention, theenzymatic activity was measured by conducting an enzymatic reactionusing agarose L03 (Takara Shuzo, Code: 5003) as a substrate and thenquantifying the resulting agarotetraose using high-performance liquidchromatography. The procedure is described in detail below.

A solution containing agarose L03 at a concentration of 0.2% in 10 mMtris-hydrochloride (pH 7.0), 10 mM calcium chloride and 10 mM sodiumchloride was prepared. 180 μl of this solution was mixed with 20 μl ofan enzyme solution. The mixture was reacted at 42° C. for 30 to 120minutes, preferably 60 minutes, and then heated at 60° C. for 1 minuteto stop the reaction. 30 μl of the reaction mixture was subjected to aTSKgel α-2500 column (inner diameter: 7.8 mm; length: 300 mm; Tosoh,Code: 18339). A peak was eluted at retention time of about 26 minutesusing 70% acetonitlrile solution as an eluent at a flow rate of 0.8ml/minute. Agarotetraose produced as a result of the enzymatic reactionin the peak was quantified. One unit (1 U) of the α-agarase of thepresent invention is defined as the amount of the enzyme that produces 1micromole of agarotetraose in 10 minutes.

100 ml of artificial seawater (product name: Jamarine S; JamarineLaboratory) was prepared. Peptone (DIFCO, Code: 0123-17-3) and yeastextract (DIFCO, Code: 0127-17-9) were added thereto at concentrations of0.3% and 0.02%, respectively. The pH was then adjusted to 8.0 using 3Msodium carbonate. The resulting mixture was transferred into a 500-mlErlenmeyer flask. 0.1 g of agar (Nacalai Tesque, Code: 010-28) was addedthereto. After sterilization in an autoclave, the microorganismTKR1-7AGα was inoculated into the mixture, and cultured at 25° C. at 120rpm overnight. The resulting culture was used as a preculture.

The main cultivation was conducted as follows. 3 l of theabove-mentioned medium was prepared in a 5-1 jar fermentor vessel andsterilized in an autoclave. 30 ml of the preculture was inoculated intothe medium and cultured at 25° C. at 250 rpm for 24 hours. The culturewas then centrifuged at 8,000×g for 20 minutes to collect about 3 l of asupernatant from which cells had been removed.

The following procedures were carried out at 4° C. or below.

The supernatant was placed in a dialysis membrane (Sanko Junyaku, Code:UC-36-32-100) and soaked in about 500 g of polyethylene glycol over twonights to concentrate until the volume of the internal solution becameabout 100 ml, and then dialyzed against Buffer A (20 mMtris-hydrochloride (pH 7.0), 10 mM calcium chloride, 10 mM sodiumchloride) for desalting. The dialyzate was loaded onto a column (φ 2cm×9.5 cm) filled with 30 ml of strong anion-exchange resin Super Q(Tosoh, Code: 17227) which had been equilibrated with the Buffer A. Theadsorbed α-agarase was eluted using a linear gradient of 10 mM to 150 mMcalcium chloride (total elution volume: 600 ml). A fraction of about 60ml with an α-agarase activity eluted at a calcium chloride concentrationbetween 50 mM and 100 mM was collected.

This fraction was dialyzed against Buffer B (10 mM tris-hydrochloride(pH 7.0), 10 mM calcium chloride, 10 mM sodium chloride) for desalting,and then loaded onto a column (φ 2 cm×6.3 cm) filled with 20 ml of SuperQ. In this case, the adsorbed enzyme was eluted using a linear gradientof 10 mM to 1.0 M sodium chloride (total elution volume: 200 ml). Afraction of 40 ml with an α-agarase activity eluted at about 0.5 M wasobtained. This fraction was dialyzed against the Buffer B, and subjectedto a column (p 0.8 cm×5.7 cm) filled with 10 ml of DEAE-TOYOPEARL(Tosoh, Code: 007473). A fraction of about 20 ml with an α-agaraseactivity was collected using a linear gradient of 10 mM to 150 mMcalcium chloride (total elution volume: 100 ml).

The fraction was then concentrated to about 1 ml using a centrifugationultrafiltration membrane Centriprep-10 (Amicon, Code: 4304). Theconcentrate was subjected to gel filtration using a column (φ 0.8 cm×60cm) filled with Sephadex G-100 (Pharmacia, Code: 17-0060-01)equilibrated with the Buffer B to obtain a fraction of about 15 ml withan α-agarase activity. This fraction was subjected to a column (φ 0.8cm×10 cm) of 5 ml of QAE-TOYOPEARL (Tosoh, Code: 14026), and elutedusing a linear gradient of 10 mM to 150 mM calcium chloride (totalelution volume: 100 ml) to obtain an α-agarase fraction of about 4 ml.

Analysis of the thus-obtained α-agarase fraction by SDS-PAGE revealedthat the enzyme of interest was purified almost to homogeneity, and thatthe molecular weight was about 95,000. The total activity of thethus-obtained purified α-agarase preparation was 45 U. This fraction wasused as Agarase 1-7 in experiments hereinafter.

EXAMPLE 2

Production of α-Agarase from TKR4-3AGα

The microorganism TKR4-3AGα was cultured as described above for themicroorganism TKR1-7AGα in Example 1 to obtain about 3 l of a culturesupernatant.

The culture supernatant was placed in a dialysis membrane (SankoJunyaku, Code: UC-36-32-100) and soaked in about 500 g of polyethyleneglycol over two nights to concentrate until the volume of the internalsolution became about 300 ml, and then dialyzed against Buffer C (10 mMtris-hydrochloride (pH 7.0), 10 mM calcium chloride, 50 mM sodiumchloride). About two thirds of the dialyzate was loaded onto a column (φ2 cm×9.5 cm) filled with 30 ml of Super Q which had been equilibratedwith the Buffer C. The adsorbed α-agarase was eluted using a lineargradient of 10 mM to 80 mM calcium chloride (total elution volume: 400ml). A fraction of about 40 ml with an α-agarase activity eluted at acalcium chloride concentration between 40 mM and 50 mM was collected.

This fraction was concentrated using Centriprep-10 and diluted with theBuffer B for desalting, and then loaded onto a column (φ 1.5 cm×5.7 cm)filled with 10 ml of DEAE-TOYOPEARL. The adsorbed α-agarase was elutedusing a linear gradient of 10 mM to 100 mM calcium chloride (totalelution volume: 100 ml). A fraction of 9 ml with an α-agarase activityeluted at a calcium chloride concentration of about 45 mM was obtained.This fraction was concentrated using Centriprep-30 (Amicon, Code: 4306),diluted 5-fold with the Buffer B, and then subjected to a column (φ 0.8cm×4 cm) filled with 2 ml of QAE-TOYOPEARL equilibrated with the BufferB. A fraction of α-agarase adsorbed to the column was collected using alinear gradient of 10 mM to 120 mM calcium chloride (total elutionvolume: 40 ml).

Analysis of the thus-obtained α-agarase fraction by SDS-PAGE revealedthat the α-agarase was purified almost to homogeneity. The totalactivity obtained was 1.53 U. This fraction was used as Agarase 4-3 inexperiments hereinafter.

EXAMPLE 3

Examination of Various Properties of Enzymes

(1) Substrate Specificity and Action

10 μl of a buffer for reaction (10 mM tris-hydrochloride (pH 7.0), 10 mMcalcium chloride, 10 mM sodium chloride) containing one selected fromthe group consisting of four types of agarooligosaccharides (agarobiose,agarotetraose, agarohexaose and agarooctaose) (each at a concentrationof 2.5 mM), three types of neoagarooligosaccharides (neoagarobiose,neoagarotetraose and neoagarohexaose) (each at a concentration of 2.5mM) and agarose L03 (at a concentration of 1%) was prepared. 10 μl of asolution of Agarase 1-7 or Agarase 4-3 was added thereto. The mixturewas reacted at 42° C. for 30 minutes. The reaction products weresubjected to thin-layer chromatography using a developing solvent havinga composition of chloroform:methanol:acetic acid =3 3:1. Afterdevelopment, reaction products were confirmed by orcinol-sulfuric acidmethod.

As a result, it was demonstrated that both Agarase 1-7 and Agarase 4-3cleave agarohexaose, agarooctaose, neoagarohexaose and agarose.

The results of thin-layer chromatography obtained when Agarase 1-7 wasallowed to act on agarohexaose are shown in FIG. 1. The results ofthin-layer chromatography obtained when Agarase 4-3 was allowed to acton agarohexaose are shown in FIG. 2. In FIGS. 1 and 2, the samplesdeveloped in the respective lanes are as follows: lane 1: agarobiose;lane 2: agarotetraose; lane 3: agarohexaose; lane 4: agarohexaosereacted with the agarase of the present invention; lane 5: a mixture ofagarobiose and agarotetraose. As seen from these figures, the α-agaraseof the present invention digests agarohexaose to generate agarobiose andagarotetraose.

(2) Identification of Product of Reaction with α-Agarase

Agarase 1-7 or Agarase 4-3 was added to 2.0 ml of a solution containingagarose L03 at a concentration of 1.0% in 10 mM tris-hydrochloride (pH7.0), 10 mM calcium chloride and 10 mM sodium chloride. The mixture wasreacted at 42° C. for 60 minutes. After reaction, a portion of thereaction mixture was subjected to a gel filtration column (Tosoh, TSKgelα-2500) and chromatographed using 70% acetonitrile as an eluent at aflow rate of 0.8 ml/minute. A fraction eluted at retention time of about26 minutes was collected and concentrated to dryness using a rotaryevaporator. The weight of the product was determined to be about 4 mg.

Analysis of the fraction by nuclear magnetic resonance using JNM-EX270FT NMR system (Nippon Denshi) confirmed that the substance which wasgenerated from agarose by the action of each enzyme and contained in thefraction was agarotetraose. Thus, it was demonstrated that both Agarase1-7 and Agarase 4-3 hydrolyzes the α-1,3 bond between D-galactose and3,6-anhydro-L-galactose in a agarose molecule to generateagarooligosaccharides containing agarotetraose.

(3) Reaction pH

10 μl of a reaction mixture containing 1% agarose L03, 10 mM calciumchloride and 10 mM sodium chloride whose pH was adjusted to thefollowing value using a buffer indicated in parentheses at a finalconcentration of 10 mM was prepared: 4.5 (acetate buffer); 5.5 (malatebuffer); 6.0, 6.5 (acetate buffer); or 7.0, 7.5 or 8.8 (tris buffer). 10μl of an enzyme solution was added thereto. The mixture was reacted at42° C. for 1 hour. The reaction mixture was subjected to thin-layerchromatography and developed using chloroform:methanol: aceticacid=3:3:1 (v/v/v) Reaction products were confirmed by orcinol-sulfuricacid method. As a result, it was demonstrated that Agarase 1-7 andAgarase 4-3 exhibit their activities of digesting agarose under neutralto weakly acidic conditions and under weakly alkaline to weakly acidicconditions, respectively.

(4) Optimal Temperature

The enzymatic activities of Agarase 1-7 and Agarase 4-3 were measured atvarious temperatures. For each of Agarase 1-7 and Agarase 4-3, thetemperature at which inactivation of the enzyme was suppressed and thereaction rapidly proceeded was 37 to 42° C.

(5) Heat Stability

A purified enzyme solution of Agarase 1-7 or Agarase 4-3 was heated at48° C., 50° C. or 60° C. for 30 seconds.

A solution containing agarose L03 at a concentration of 0.2% in 10 mMtris-hydrochloride (pH 7.0), 10 mM calcium chloride and 10 mM sodiumchloride was then added to the heated solution. The mixture was reactedat 42° C. for 1 hour. The reaction was terminated by heating for 1minute in boiling water. Reaction products were quantified according tothe method for measuring an activity as described in Example 1. As aresult, Agarase 1-7 exhibited 25% of its activity after treatment at 48°C., and Agarase 4-3 exhibited 22% of its activity after treatment at 50°C., defining the activity without the heat treatment as 100%.

(6) Molecular Weight

The molecular weight of Agarase 1-7 was determined by SDS-polyacrylamidegel electrophoresis. Agarase 1-7 was electrophoresed using a 10-20%polyacrylamide gradient gel containing SDS along with a molecular weightmarker (Bio-Rad, myosin (m.w. 200,000), β-galactosidase (m.w. 116,250),phosphorylase b (m.w. 97,400), bovine serum albumin (m.w. 66,200),ovalbumin (m.w. 45,000), carbonic anhydrase (m.w. 31,000), trypsininhibitor (m.w. 21,500), lysozyme (m.w. 14,400)). The molecular weightof Agarase 1-7 was about 95,000 dalton as determined based on themobility on the gel.

The molecular weight of Agarase 4-3 was determined by equilibriumdensity-gradient centrifugation. Density gradient was prepared byvarying the concentration of glycerol from 15% to 35% in a buffercontaining 10 mM tris-HCl (pH 7.0), 10 mM calcium chloride and 50 mMsodium chloride. 4.8 ml of linear density gradients were prepared in two5-ml centrifugation tubes such that the lowermost layer containedglycerol at a concentration of 35% and the uppermost layer containedglycerol at a concentration of 15%. 100 μl of a solution containing 15μl of a molecular weight marker Low Range (Bio-Rad) and glycerol at aconcentration of 15% in the above-mentioned buffer was overlaid onto thetop of one of the tubes. 100 μl of the enzyme preparation of Agarase 4-3was overlaid onto the other tube. These centrifugation tubes werecentrifuged using a swing rotor at 45,000 rpm at 4° C. for 21 hours.After centrifugation, fractions 1 to 20 each containing 250 μl of thebuffer were collected from the top to the bottom of each centrifugationtube. The respective fractions collected from the centrifugation tube towhich the molecular weight marker was added were subjected to SDS-PAGE.The respective fractions collected from the centrifugation tube to whichthe enzyme preparation was added were subjected to SDS-PAGE andmeasurement of enzymatic activities. A peak of the enzymatic activity ofα-agarase was detected for the fraction nos. 8-10 corresponding tomolecular weight of about 65,000 to 85,000 based on the results ofSDS-PAGE of the fractions containing the molecular weight marker. Basedon these results as well as the results of SDS-PAGE of the fractionscontaining the enzyme preparation, the molecular weight of Agarase 4-3was estimated to be about 85,000.

(7) Amino Acid Sequence Analysis by Edman Degradation Method

The amino acid sequences of α-Agarase 1-7 and α-Agarase 4-3 obtained inExamples 1 and 2 were determined by Edman degradation method. A purifiedenzyme preparation containing Agarase 1-7 or Agarase 4-3 correspondingto 10 pmole of the enzyme protein was subjected to SDS-PAGE using a10-20% polyacrylamide gradient gel. After electrophoresis, the enzymeseparated on the gel was blotted onto a membrane ProBlot (AppliedBiosystems). A portion of the membrane to which the enzyme had beenadsorbed was analyzed using a protein sequencer G100A (Hewlett Packard).As a result, an amino acid sequence P1:Asp-Thr-Leu-Ser-Val-Glu-Ala-Glu-Met-Phe (SEQ ID NO: 1) and an amino acidsequence P2:Gly-Asp-Ile-Val-Ile-Glu-Leu-Glu-Asp-Phe-Asp-Ala-Thr-Gly-Thr-Thr-Gly-Arg-Val-Ala(SEQ ID NO:2) were determined for Agarase 1-7 and Agarase 4-3,respectively.

EXAMPLE 4

Production of Agarooligosaccharides

Agarose L03 was added to 2 ml of a buffer for reaction (10 mMtris-hydrochloride (pH 7.0), 10 mM calcium chloride, 10 mM sodiumchloride) at a concentration of 1.0% (w/v). 2 U of the purifiedpreparation of Agarase 1-7 was further added thereto. The mixture wasreacted at 42° C. for 16 hours. After reaction, the reaction mixture wasfiltered through a 0.22-μm filter (Millipore, Code: SLGVL040S). Thefiltered reaction mixture was analyzed using high-performance liquidchromatography under the same conditions as those used for measuring theactivity of the enzyme of the present invention in Example 1 todetermine the generated agarooligosaccharides. As a result, agarobiose,agarotetraose and agarohexaose were detected in the reaction mixture.Thus, it was confirmed that the above-mentioned enzymatic reactionproduces these smaller agarooligosaccharides.

EXAMPLE 5

Preparation of Chromosomal DNAs from TKR1-7AGα and TKR4-3AGα

Artificial seawater (product name: Jamarine S; Jamarine Laboratory) wasprepared. Peptone (DIFCO, Code: 0123-17-3) and yeast extract (DIFCO,Code: 0127-17-9) were added thereto at concentrations of 0.3% (w/v) and0.02% (w/v), respectively,. The pH was then adjusted to 8.0 using 3Msodium carbonate. Agar (Nacalai Tesque, Code: 010-28) was added theretoat a concentration of 0.1% (w/v). The mixture was sterilized in anautoclave. 10 μl of a glycerol stock of one of the α-agarase-producingstrains TKR1-7AGα and TKR4-3AGα was inoculated into 2 ml of the mediumand cultured at 25° C. overnight. 1 ml of the culture was inoculatedinto 100 ml of the same medium and cultured at 25° C. overnight. Thecells were collected by centrifugation at 8,000×g for 10 minutes. Thecells were suspended in 10 ml of Buffer A (100 mM sodium chloride, 100mM tris-hydrochloride (pH-8.0), 10 mM EDTA (pH 8.0)). 0.25 ml of alysozyme solution (20 mg/ml) was added thereto. The mixture wasincubated at 37° C. for 1 hour. 2.5 ml of the Buffer A containing SDS ata concentration of 5% was then added thereto. The resulting mixture wasincubated at 60° C. for 20 minutes while shaking. 1.5 ml of a protease Ksolution (20 mg/ml) was added thereto. The mixture was incubated at 37°C. overnight. Almost equal volume of phenol was then added thereto, andthe mixture was gently shaken at room temperature for about 10 minutes.The mixture was centrifuged at 2,000×g for 10 minutes. The supernatantwas transferred into cold ethanol. A chromosomal DNA was wound using aglass bar. After repeating the procedure twice, 50 μl of an RNasesolution (10 mg/ml) was added, and the mixture was incubated at 37° C.for 10 minutes. The chromosomal DNA was recovered from the solution byethanol precipitation and suspended in 5 ml of Buffer, B (140 mM sodiumchloride, 20 mM tris-hydrochloride (pH 7.5), 1 mM EDTA (pH 7.5)). Thesuspension was dialyzed against the same buffer overnight. Then, about1.5 mg and about 3.1 mg of chromosomal DNAs were obtained from TKR1-7AGαand TKR4-3AGα, respectively. The purity of each DNA was examined basedon OD260 nm/280 nm. The value was about 1.8 in each case. Thechromosomal DNAs were used for cloning as described below.

EXAMPLE 6

Cloning of α-Agarase 1-7 Gene

10 μg of the chromosomal DNA from TKR1-7AGα partially digested with arestriction enzyme BamHI was subjected to electrophoresis using 1.0% lowmelting point agarose gel. After staining with ethidium bromide, aportion corresponding to 4 to 10 kb was excised under ultravioletirradiation. The DNA was extracted and purified from the gel byheat-melting according to a conventional method. The recovered DNAfragments were inserted into a BamHI site in a plasmid pUC19 (TakaraShuzo) using a DNA ligation kit (Takara Shuzo). The plasmid library wasused to transform Escherichia coli JM109. Transformants were grown on LBagar medium containing agar at a concentration of 1.5% (w/v) andampicillin at a concentration of 50 μg/ml. After culturing at 37° C.overnight, about 1000 transformants were grown on one plate. 20 μlateswere further incubated at 25° C. for 4 days. As a result, degradation ofsurrounding agar was observed for two colonies. Each of these strainswas inoculated into 2 ml of LB medium containing ampicillin and culturedat 37° C. overnight. An α-agarase activity was recognized for a crudeextract prepared from the resulting cells. A plasmid DNA was extractedfrom each transformant according to a conventional method. The length ofthe inserted DNA was determined by digestion with a restriction enzymeBamHI to be about 7.2 kb for each of the transformants. These hybridplasmids were considered to be identical each other based on therestriction enzyme digestion patterns, and designated as pAA1.Escherichia coli transformed with the plasmid pAA1 is designated andindicated as Escherichia coli JM109/pAA1, and deposited under BudapestTreaty on May 26, 1999 (the date of the original deposit) at theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry under accession number FERM BP-6992.

A mixed primer 1 represented by SEQ ID NO: 3 was designed based on theamino acid sequence of Agarase 1-7, P1, represented by SEQ ID NO:1. APCR was carried out using this primer and a primer 2 represented by SEQID NO: 4 which specifically hybridizes to a pUC vector as well as pAA1as a template.

50 ng of pAA1, 5 μl of ExTaq buffer, 8 μl of a dNTP mixture, 1 μl of themixed primer 1, 1 μl of the primer 2 and 0.5 μl of TaKaRa ExTaq wereadded to a 0.5-ml tube for PCR. Sterile water was further added theretoto make the total volume to 50 μl. After the solution was overlaid with50 μl of mineral oil, the tube was placed in an automated geneamplification instrument Thermal Cycler (Takara Shuzo). After denaturingat 94° C. for 2 minutes, a PCR of 30 cycles was performed. Each cycleconsisted of denaturation at 94° C. for 1 minute, annealing of primersat 50° C. for 2 minutes and synthesis reaction at 72° C. for 3 minutes.After the PCR, the whole reaction mixture was subjected toelectrophoresis on 1.0% low melting point agarose gel. An amplified DNAfragment of about 3.5 kb excised from the gel was ligated to pT7Blue(Novagen). The nucleotide sequence of this DNA fragment was determinedfrom the N-terminal side according to the dideoxy chain terminatormethod using Taq DNA polymerase. A primer represented by SEQ ID NO: 5was designed based on the sequence determined as described above. Theupstream nucleotide sequence was determined using this primer. As aresult, it was found that there exists a nucleotide sequence thatencodes 27 amino acids upstream from the nucleotide sequencecorresponding to the amino acid sequence P1 represented by SEQ ID NO: 1,there exists an SD-like sequence in the further upstream region, andpAA1 contains an open reading frame (ORF) that encodes a polypeptideconsisting of a total of 925 amino acids. The nucleotide sequence of theopen reading frame and the amino acid sequence encoded by the openreading frame are shown in SEQ ID NOS: 12 and 14, respectively. TheN-terminal sequence of the α-agarase purified from the microorganismTKR1-7AGα, P1, matched with the sequence of the 28th to 37th amino acidsin the amino acid sequence of SEQ ID NO: 13.

EXAMPLE 7

Cloning of α-Agarase 4-3 Gene

Mixed primers 3 and 4 were designed based on the sequences of the aminoacid numbers 2-11 and 12-20 in the amino acid sequence of Agarase 4-3,P2, represented by SEQ ID NO: 2. These primers were used to clone a genefor Agarase 4-3 using LA PCR in vitro cloning kit (Takara Shuzo). Thesequences of the mixed primers 3 and 4 are shown in SEQ ID NOS: 6 and 7,respectively.

A primary PCR was carried out as follows. The chromosomal DNA preparedfrom TKR4-3AGα in Example 5 was completely digested with a restrictionenzyme BamHI. BamHI linkers were ligated to the termini of the digestedDNA using a DNA ligation kit (Takara Shuzo). A portion of the ligationmixture was placed in a 0.5-ml tube for PCR, and 5 μl of 10×LA PCRbuffer, 8 μl of a dNTP mixture, 1 μl of the mixed primer 3, 1 μl of aprimer C1 attached to LA PCR in vitro cloning kit (Takara Shuzo) and 0.5μl of TaKaRa LATaq were added thereto. Sterile water was further addedthereto to make the total volume to 50 μl. After the solution wasoverlaid with 50 μl of mineral oil, the tube was placed in an automatedgene amplification instrument Thermal Cycler (Takara Shuzo). Afterdenaturing at 94° C. for 2 minutes, a PCR of 30 cycles was performed.Each cycle consisted of denaturation at 94° C. for 1 minute, annealingof primers at 50° C. for 2 minutes and synthesis reaction at 72° C. for3 minutes.

A secondary PCR was carried out using the thus-obtained product of theprimary PCR. A PCR was carried out using 1 μl of the reaction mixtureafter the primary PCR as a template as well as a combination of themixed primer 4 and a primer C2 attached to LA PCR in vitro cloning kit(Takara Shuzo) under the same conditions as those used for the primaryPCR. After removing the overlaid mineral oil, 5 μl of the reactionmixture was subjected to electrophoresis on 1.0% agarose gel. Anamplification product was confirmed by staining DNA with ethidiumbromide. As a result, an amplification product of about 2 kb wasobserved and the DNA fragment was designated as 4-3N.

This amplified fragment was excised from the agarose gel, extracted andpurified according to a conventional method and ligated to a vectorpT7Blue. The ligation mixture was used to transform Escherichia coliJM109. The nucleotide sequences of the terminal regions of 4-3 weredetermined according to the dideoxy chain terminator method using theresulting transformants.

Primers were designed based on the determined sequences of the terminalregions of 4-3N. DNA fragments located upstream and downstream from 4-3Nwere cloned using LA PCR in vitro cloning kit (Takara Shuzo).

Primers 5 and 6 represented by SEQ ID NOS: 8 and 9, respectively,designed based on the sequence of the region around the N-terminus amongthe terminal regions of 4-3N determined as described above were used forcloning a DNA fragment located upstream from 4-3N. Cloning was carriedout as described above for the cloning of 4-3N except that thetemperature for annealing of primers was changed to 55° C. As a result,a DNA fragment of about 1.0 kb was obtained and designated as 4-3UN.

Primers 8 and 9 represented by SEQ ID NOS: 10 and 11, respectively,designed based on the sequence of the region around the C-terminus amongthe terminal regions of 4-3N determined as described above were used forcloning a DNA fragment located downstream from 4-3N. Cloning was carriedout as described above for the cloning of 4-3N except that thetemperature for annealing of primers was changed to 55° C. As a result,a DNA fragment of about 2.0 kb was obtained and designated as 4-3C.

Each of the thus-obtained fragments 4-3UN and 4-3C was ligated to avector pT7Blue (Novagen). The nucleotide sequences were determinedaccording to the dideoxy chain terminator method. Analysis and alignmentof the nucleotide sequences of the DNA fragments 4-3N, 4-3UN and 4-3Cdetermined as described above revealed an open reading frame (ORF) thatencodes a polypeptide consisting of 951 amino acids. The nucleotidesequence of the open reading frame and the amino acid sequence encodedby the nucleotide sequence of the open reading frame are shown in SEQ IDNOS: 13 and 15, respectively. In 4-3UN, a nucleotide sequence for theamino acid sequence P2, an upstream nucleotide sequence encoding 183amino acids, and a further upstream SD-like sequence were found. TheN-terminal amino acid sequence determined for the α-agarase purifiedfrom the microorganism TKR4-3, P2, matched with the sequence of the184th to 203rd amino acids in the amino acid sequence of SEQ ID NO: 15.

The amino acid sequences of Agarase 1-7 and Agarase 4-3 obtained asdescribed above as well as the nucleotide sequences of the genes have nohomology with the amino acid sequences of β-agarases, which are knownagarose-digesting enzymes that cleave agarose in a different manner fromthe agarase of the present invention, and the nucleotide sequences ofthe genes. Thus, these sequences are considered to be absolutely novel.

EXAMPLE 8

Construction of Plasmid for Expressing α-Agarase 1-7

A primer 10 having a sequence of SEQ ID NO: 18 was synthesized. Theprimer 10 is a primer that has a recognition sequence for a restrictionenzyme NdeI at base numbers 8 to 13 and a nucleotide sequencecorresponding to an amino acid sequence of amino acid numbers 28 to 31in the amino acid sequence of α-Agarase 1-7 (SEQ ID NO: 14) at basenumbers 14 to 25.

A PCR was carried out using the primer 10 and the primer 2 (SEQ ID NO:4)-which hybridizes to a portion of the vector pUC19 in pAA1 as well aspAA1 as a template.

10 pmol each of the primer 10 and the primer 2, 10 ng of pAA1 obtainedin Example 6 as a template, 5 μl of 10× ExTaq buffer, 8 μl of a dNTPmixture and 0.5 μl of TaKaRa ExTaq were added to a 0.5-ml tube for PCR.Sterile water was further added thereto to make the total volume to 50μl. The tube was placed in an automated gene amplification instrumentThermal Cycler (Takara Shuzo). After denaturing at 94° C. for 2 minutes,a PCR of 25 cycles was performed. Each cycle consisted of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes. The PCR productwas concentrated and desalted by ethanol precipitation, doubly digestedwith restriction enzymes NdeI (Takara Shuzo) and BamHI (Takara Shuzo),and then subjected to electrophoresis on 1.0% agarose gel. TheNdeI-BamHI digest was separated, extracted and purified. The purifiedproduct was mixed with and ligated to pKF19k (Takara Shuzo) digestedwith NdeI and BamHI using a DNA ligation kit (Takara Shuzo). 10 μl ofthe ligation mixture was used to transform Escherichia coli JM109.Transformants were grown on LB medium containing agar at a concentrationof 1.5% (w/v) and kanamycin at a concentration of 50 μg/ml. Plasmidswere prepared from white colonies, DNA sequencing was carried out, and aplasmid into which the PCR product was properly inserted was selectedand designated as pAA201. pAA201 is a plasmid that encodes an amino acidsequence of amino acid numbers 28 to 925 in the amino acid sequence ofα-Agarase 1-7 (SEQ ID NO:14).

The transformant having pAA201 being introduced was inoculated into 2.5ml of LB broth containing 50 μg/ml kanamycin and 10 mM calcium chlorideand cultured at 37° C. overnight. A portion of the culture wasinoculated into 2.5 ml of the same fresh medium and cultured at 25° C.until it reached exponential growth phase. At that time, IPTG was addedthereto at a final concentration of 1.0 mM. The cultivation wascontinued at 15° C. overnight to induce the expression of the protein ofinterest. The cells were then collected by centrifugation andresuspended in 150 μl of a cell destruction solution (20 mMtris-hydrochloride (pH 7.0), 10 mM calcium chloride, 10 mM sodiumchloride). The cells were destroyed by sonication. An extract as asupernatant and a precipitate were separated each other bycentrifugation and used as samples for the measurement of α-agaraseactivities using agarose as a substrate. Then, an activity was observedfor the extract. The activity contained in 100 ml of the culture wasabout 25-fold higher than that of the wild type strain TKR1-7AGα.

EXAMPLE 9

Expression System for α-Agarase 1-7 Using pT7BlueT Vector

A primer 11 having a sequence of SEQ ID NO: 19 was synthesized. Theprimer 11 is a primer that has a nucleotide sequence corresponding to anamino acid sequence of amino acid numbers 28 to 33 in the amino acidsequence of α-Agarase 1-7 (SEQ ID NO: 14) at base numbers 13 to 30.

A PCR was carried out using the primer 11 and the primer 2 as well aspAA1 as a template. The PCR product was separated by electrophoresis on1.0% agarose gel, extracted and purified. The purified PCR product wasligated to pT7BlueT (Takara Shuzo), a vector designed for cloning, toconstruct an expression vector. The conditions used for the PCR werethose as described in Example 8. DNA sequencing was carried out and aplasmid into which the PCR product was properly inserted was selected.

The selected hybrid plasmid was designated as pAA301. pAA301 is aplasmid that encodes an amino acid sequence of amino acid numbers 28 to925 in the amino acid sequence of α-Agarase 1-7 (SEQ ID NO:14) likepAA201.

pAA301 was used to transform Escherichia coli JM109 and the resultingtransformant was inoculated into LB medium containing 50 μg/mlampicillin and 10 mM calcium chloride. The expression of the protein ofinterest was induced using IPTG, and the α-agarase activity wasdetermined as described above in Example 8. Then, an activity wasobserved for the extract. The activity contained in 100 ml of theculture was about 100-fold higher than that of TKR1-7AGα.

EXAMPLE 10

Expression System for α-Agarase 1-7 Using pET16b

A primer 12 having a sequence of SEQ ID NO: 20 was synthesized. Theprimer 12 is a primer that has a nucleotide sequence complementary to anucleotide sequence corresponding to amino acid numbers 919 to 924 inthe amino acid sequence of α-Agarase 1-7 (SEQ ID NO: 14) at base numbers17 to 34 and a recognition sequence for a restriction enzyme BamHI atbase numbers 9 to 14.

A PCR was carried out using the primer 10 (SEQ ID NO: 18) and the primer12 (SEQ ID NO: 20) as well as pAA1 as a template under conditions asdescribed in Example 8. The amplified fragment was concentrated anddesalted by ethanol precipitation, digested with NdeI (Takara Shuzo) andBamHI (Takara Shuzo), separated by agarose gel electrophoresis,extracted and purified. The product was ligated to pET16b (Takara Shuzo)digested with NdeI and BamHI. The resulting hybrid plasmid wasdesignated as pAA401. pAA401 is a plasmid that encodes an amino acidsequence of amino acid numbers 28 to 925 in the amino acid sequence ofα-Agarase 1-7 (SEQ ID NO:14) like pAA201 and pAA301.

pAA401 was used to transform Escherichia coli BL21(DE3)pLysS and theresulting transformant was used to determine the α-agarase activity asdescribed above in Example 8 except that ampicillin was used as a drugin place of kanamycin. Then, an activity was observed for the extract.The activity contained in 100 ml of the culture was about 100-foldhigher than that of TKR1-7AGα.

EXAMPLE 11

Construction of Plasmid for Expressing α-Agarase 4-3

A primer 13 having a sequence of SEQ ID NO: 21 and a primer 14 having asequence of SEQ ID NO: 22 were synthesized.

The primer 13 is a primer that has a recognition sequence for arestriction enzyme NdeI at base numbers 12 to 17 and a nucleotidesequence corresponding to an amino acid sequence of amino acid numbers184 to 187 in the amino acid sequence of α-Agarase 4-3 (SEQ ID NO: 15)at base numbers 18 to 29.

The primer 14 is a primer that has a recognition sequence for arestriction enzyme BamHI at base numbers 8 to 13 and a sequence thathybridizes to a region downstream from the open reading frame of theα-Agarase 4-3 gene obtained by cloning.

A PCR was carried out using the primers 13 and 14 as well as thechromosomal DNA from the wild type strain TKR4-3AGα as a template. Areaction mixture for PCR containing 10 pmol each of the primers 13 and14, 10 ng of the chromosomal DNA from the wild type strain TKR4-3AGαdigested with a restriction enzyme BamHI as a template and ExTaq (TakaraShuzo) was prepared. After denaturing at 94° C. for 2 minutes, a PCR of25 cycles was performed. Each cycle consisted of 94° C. for 1 minute,50° C. for 2 minutes and 72° C. for 3 minutes. The resulting PCR productwas concentrated by ethanol precipitation and doubly digested withrestriction enzymes NdeI (Takara Shuzo) and BamHI (Takara Shuzo). TheNdeI-BamHI digest was separated by electrophoresis on 1.0% agarose gel,extracted and purified. A hybrid plasmid with pKF19k was constructed asdescribed in Example 8 and transformation of Escherichia coli JM109 wascarried out. Plasmids were prepared from the resulting transformants,and a plasmid into which the PCR product was inserted in a properdirection was selected and designated as pAH101. pAH101 is a plasmidthat encodes an amino acid sequence of amino acid numbers 184 to 951 inthe amino acid sequence of α-Agarase 4-3 (SEQ ID NO:15).

Escherichia coli transformed with the plasmid pAH101 is designated andindicated as Escherichia coli JM109/pAH101, and deposited under BudapestTreaty on Jan. 27, 2000 at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki, Japan under accession number FERM BP-7008.

The α-agarase activity was determined for the transformant harboringpAH101 as described above in Example 8. Then, an activity was observedfor the extract. The activity contained in 100 ml of the culture wasabout 15-fold higher than that of the wild type strain TKR4-3AGα.

EXAMPLE 12

Expression System for α-Agarase 4-3 Using pET16b

A primer 15 having a sequence of SEQ ID NO: 23 was synthesized.

The primer 15 is a primer that has a recognition sequence for arestriction enzyme BamHI at base numbers 10 to 15 and a nucleotidesequence complementary to a nucleotide sequence corresponding to anamino acid sequence of amino acid numbers 945 to 950 in the amino acidsequence of α-Agarase 4-3 (SEQ ID NO: 15) at base numbers 18 to 35.

A PCR was carried out using the primers 13 and 15 as well as thechromosomal DNA from the wild type strain TKR4-3AGα described in Example11 as a template under conditions as described in Example 8. Theamplified fragment was concentrated by ethanol precipitation, digestedwith NdeI and BamHI, separated by agarose gel electrophoresis, extractedand purified. The product was ligated to pET16b (Takara Shuzo) digestedwith NdeI and BamHI. The resulting hybrid plasmid was designated aspAH201. pAH201 is a plasmid that encodes an amino acid sequence of aminoacid numbers 184 to 950 in the amino acid sequence of α-Agarase 4-3 (SEQID NO:15).

pAH201 was used to transform Escherichia coli BL21(DE3)pLysS and theresulting transformant was used to determine the α-agarase activitydescribed above in Example 8 except that ampicillin was used as a drugin place of kanamycin. Then, an activity was observed for the extract.The activity contained in 100 ml of the culture was about 75-fold higherthan that of TKR4-3AGα.

EXAMPLE 13

Activity of Modified Protein

A modified protein was prepared by means of genetic engineering asdescribed below. The α-agarase activity of the modified protein wasdetermined.

A primer 16 having a sequence of SEQ ID NO: 24 was synthesized.

The primer 16 is a primer that has a nucleotide sequence correspondingto an amino acid sequence of amino acid numbers 172 to 174 in the aminoacid sequence of α-Agarase 1-7 (SEQ ID NO: 14) at base numbers 3 to 11,a nucleotide sequence corresponding to an amino acid sequence of aminoacid numbers 177 to 181 in the amino acid sequence of α-Agarase 1-7 (SEQID NO: 14) at base numbers 18 to 32 and a recognition sequence for arestriction enzyme NdeI at base numbers 12 to 17.

A PCR was carried out using the primer 16 and the primer 2 (SEQ ID NO:4) as well as pAA1 as a template. The product was ligated to pKF19k, andtransformation of Escherichia coli JM109 was carried out as described inExample 8. Plasmids were prepared from the resulting transformants. Ahybrid plasmid obtained by confirming the DNA sequence at the connectionsite was designated as pAA501. pAA501 is a plasmid that encodes apolypeptide in which a portion up to amino acid number 176 in α-Agarase1-7 is deleted, i.e., an amino acid sequence of amino acid numbers 177to 925 in SEQ ID NO:14.

Escherichia coli JM109 transformed with pAA501 was cultured as describedin Example 8 and the expression of the protein encoded by pAA501 wasinduced using IPTG, and the activity was determined. Then, an α-agaraseactivity was observed for the extract.

EXAMPLE 14

Southern Hybridization

Southern hybridization was carried out against the chromosomal DNA fromthe wild type strain TKR1-7AGα using the fragment inserted in anα-Agarase 4-3 clone pAH101 as a probe. The following procedure wascarried out according to the protocol for DIG DNA labeling/detection kit(Roche). In this case, pAH101 was digested with restriction enzymes NdeIand BamHI. The resulting DNA fragment of about 2.4 kb was separated byagarose gel electrophoresis, extracted and purified. About 1.0 μg of thepurified fragment was labeled according to the protocol for theabove-mentioned kit and used as a probe.

About 2.0 μg of the chromosomal DNA from TKR1-7AGα was digested withBamHI and electrophoresed on 1.0% agarose gel. The DNA fragments weretransferred onto a nitrocellulose membrane (Amersham) using 0.4N sodiumhydroxide according to the conventional method. Prehybridization wasthen carried out at 68° C. for 1 hour. The labeled probe washeat-denatured and added thereto. Hybridization was carried out at 68°C. overnight in a solution containing 6×SSC, 0.5% SDS, 5× Denhardt's and100 mg/ml herring sperm DNA. The membrane was washed twice in 2×SSC,0.1% (w/v) SDS for 5 minutes at room temperature followed by twice in0.1×SSC, 0.1% (w/v) SDS for 15 minutes at 68° C. to eliminate excessprobe. A detection reaction was then carried out according to theprotocol. As a result, a band was observed at a position correspondingto about 7.2 kb. As described above, a gene encoding an activeα-agarase, the α-agarase gene from the wild type strain TKR1-7AGα, couldbe detected by conducting hybridization using the α-Agarase 4-3 gene asa probe under stringent conditions. Comparison of the entire sequencesof the open reading frames for Agarase 1-7 and Agarase 4-3 revealed thatthey share homologies in the amino acid sequence and the nucleotidesequence of the gene of 51% and 61%, respectively.

EXAMPLE 15

Preparation of Deleted Forms of α-Agarase

Proteins each having deletion at N-terminus as described below were madeby means of genetic engineering, and the α-agarase activities for therespective proteins were determined.

(1) α-Agarase Activities of Deleted Forms of α-Agarase 1-7

A primer 17 (SEQ ID NO:25) has nucleotide sequences corresponding toamino acid sequences of amino acid numbers 197 to 200 and 201 to 206 inthe amino acid sequence of α-Agarase 1-7 (SEQ ID NO: 14) at base numbers1 to 12 and 19 to 36 and a recognition sequence for a restriction enzymeNdeI at base numbers 13 to 18. A PCR was carried out using this primerand the primer 2 (SEQ ID NO: 4) as well as the plasmid pAA1 as atemplate as follows. 10 pmol each of the primer 17 and the primer 2, 10ng of pAA1 as a template, 5 μl of 10× ExTaq buffer, 8 μl of a dNTPmixture and 0.5 μl of TaKaRa ExTaq were added to a 0.5-ml tube for PCR.Sterile water was further added thereto to make the total volume to 50μl. The tube was placed in an automated gene amplification instrumentThermal Cycler (Takara Shuzo). After denaturing at 94° C. for 2 minutes,a PCR of 25 cycles was performed. Each cycle consisted of 94° C. for 1minute, 55° C. for 2 minutes and 72° C. for 3 minutes. The PCR productwas concentrated and desalted by ethanol precipitation, and doublydigested with restriction enzymes NdeI (Takara Shuzo) and BamHI (TakaraShuzo). The digest was subjected to electrophoresis on 1.0% agarose gel.The PCR product digested with NdeI and BamHI was extracted and purified.The purified PCR product was mixed with and ligated to pKF19k (TakaraShuzo) digested with NdeI and BamHI using a DNA ligation kit (TakaraShuzo). 10 μl of the ligation mixture was used to transform Escherichiacoli JM109. Transformants were grown on LB medium containing agar at aconcentration of 1.5% (w/v) and kanamycin at a concentration of 50μg/ml. Plasmids were prepared from white colonies, nucleotide sequencesof the fragments inserted into the plasmids were determined, and aplasmid into which the PCR product was inserted was selected anddesignated as pAA-deN1. The protein expressed from this plasmid is onein which a portion up to amino acid number 200 is deleted in α-Agarase1-7.

A transformant obtained by introducing pAA-deN1 into Escherichia coliwas inoculated into 2.5 ml of LB broth containing 50 μg/ml kanamycin and10 mM calcium chloride and cultured at 37° C. overnight. A portion ofthe culture was inoculated into 2.5 ml of the same fresh medium andcultured at 25° C. until it reached exponential growth phase. At thattime, IPTG was added thereto at a final concentration of 1.0 nM. Thecultivation was continued at a lower cultivation temperature, 15° C.,overnight to induce the expression of the protein of interest. The cellswere then collected by centrifugation and suspended in 150 μl of a celldestruction solution (20 mM tris-hydrochloride (pH 7.0), 10 mM calciumchloride, 10 mM sodium chloride). The cells were destroyed bysonication. A cell-free extract as a supernatant and a precipitate wereseparated each other by centrifugation and used as samples for themeasurement of α-agarase activities using agarose as a substrateaccording to the method as described above. Then, an activity wasobserved for the cell-free extract.

Deleted forms were made in a similar manner and their α-agaraseactivities were determined.

A primer 18 (SEQ ID NO:26) has nucleotide sequences corresponding toamino acid sequences of amino acid numbers 332 to 334 and 335 to 340 inthe amino acid sequence of α-Agarase 1-7 (SEQ ID NO: 14) at base numbers3 to 11 and 18 to 35 and a recognition sequence for a restriction enzymeNdeI at base numbers 12 to 17. A hybrid plasmid constructed by usingthis primer in place of the primer 17 was designated as pAA-deN2. Theprotein expressed from this plasmid is one in which a portion up toamino acid number 334 is deleted in α-Agarase 1-7.

A primer 19 (SEQ ID NO:27) has nucleotide sequences corresponding toamino acid sequences of amino acid numbers 376 to 378 and 379 to 385 inthe amino acid sequence of α-Agarase 1-7 (SEQ ID NO: 14) at base numbers2 to 10 and 18 to 38 and a recognition sequence for a restriction enzymeNdeI at base numbers 11 to 16. A hybrid plasmid constructed by usingthis primer in place of the primer 17 was designated as pAA-deN3. Theprotein expressed from this plasmid is one in which a portion up toamino acid number 378 is deleted in α-Agarase 1-7. The results for theα-agarase activities expressed by transformants having these plasmidsbeing introduced are summarized in Table 2.

(2) α-Agarase Activities of Deleted Forms of α-Agarase 4-3

Deleted forms of Agarase 4-3 were made as described above for Agarase1-7.

A primer 20 (SEQ ID NO:28) has nucleotide sequences corresponding toamino acid sequences of amino acid numbers 247 to 250 and 251 to 255 inthe amino acid sequence of α-Agarase 4-3 (SEQ ID NO: 15) at base numbers1 to 12 and 19 to 33 and a recognition sequence for a restriction enzymeNdeI at base numbers 13 to 18. A PCR was carried out using this primerand the primer 2 (SEQ ID NO: 4) as well as the plasmid pAH101 describedin Example 11 as a template. The procedure as described in Example 15(1) was then carried out to construct a hybrid plasmid with pKF19k,which was designated as pAH-deN1. A protein in which a polypeptide up toamino acid number 250 is deleted in the amino acid sequence of α-Agarase4-3 (SEQ ID NO:15) is expressed from Escherichia coli JM109 having thishybrid plasmid being introduced. The activity was determined for thetransformant as described in Example 15 (1). Then, an α-agarase activitywas observed for the cell-free extract.

A primer 21 (SEQ ID NO:29) has nucleotide sequences corresponding toamino acid sequences of amino acid numbers 361 to 364 and 365 to 370 inthe amino acid sequence of α-Agarase 4-3 (SEQ ID NO: 15) at base numbers3 to 14 and 21 to 38 and a recognition sequence for a restriction enzymeNdeI at base numbers 15 to 20. A plasmid constructed by using thisprimer in place of the primer 20 was designated as pAH-deN2. A proteinin which a polypeptide up to amino acid number 364 is deleted in theamino acid sequence of α-Agarase 4-3 (SEQ ID NO:15) is expressed fromEscherichia coli JM109 having this plasmid being introduced.

A primer 22 (SEQ ID NO:30) has nucleotide sequences corresponding toamino acid sequences of amino acid numbers 406 to 408 and 409 to 413 inthe amino acid sequence of α-Agarase 4-3 (SEQ ID NO: 15) at base numbers3 to 11 and 18 to 32 and a recognition sequence for a restriction enzymeNdeI at base numbers 12 to 17. A plasmid constructed by using thisprimer in place of the primer 20 was designated as pAH-deN3. A proteinin which a polypeptide up to amino acid number 408 is deleted in theamino acid sequence of α-Agarase 4-3 (SEQ ID NO:15) is expressed fromthis plasmid being introduced. The results for the α-agarase activitiesexpressed by transformants having these plasmids being introduced aresummarized in Table 2. TABLE 2 α-Agarase activity pAA-deN1 + pAA-deN2 +pAA-deN3 − pAH-deN1 + pAH-deN2 + pAH-deN3 −

The present invention provides a novel α-agarase. It is possible toproduce agarooligosaccharides with low degrees of polymerization having3,6-anhydro-L-galactose at their reducing ends (e.g., agarobiose andagarotetraose) directly from agarose by using said enzyme. Theagarooligosaccharides produced using the α-agarase of the presentinvention have physiological activities such as an apoptosis-inducingactivity, a carcinostatic activity, various antioxidant activities, animmunoregulatory activity and an antiallergic activity. Thus, they areuseful in the fields of pharmaceutical compositions, foods and drinks.

The present invention discloses an amino acid sequence and a nucleotidesequence for an α-agarase for the first time. Thus, it is possible toprovide a gene encoding a polypeptide having an α-agarase activity. Thepresent invention also provides an industrially advantageous method forproducing a polypeptide having an α-agarase activity by geneticengineering using said gene.

Furthermore, addition of agarose to a medium for inducing the productionof the α-agarase is not required in the production method by geneticengineering using said gene. Thus, it is considered that labor can besaved upon cultivation and the enzyme is readily purified.

In addition, based on the fact that the present invention provides anα-agarase gene for the first time, the present invention provides, basedon the information on said gene, a recombinant polypeptide encoded bythe gene, an antibody that specifically binds to the polypeptide or afragment thereof, as well as a probe or a primer that specificallyhybridize to α-agarase.

Sequence Listing Free Text

SEQ ID NO:1: N-terminal amino acid sequence of agarase 1-7.

SEQ ID NO:2: N-terminal amino acid sequence of agarase 4-3.

SEQ ID NO:3: Designed oligonucleotide primer for PCR using pAA1 astemplate.

SEQ ID NO:4: Designed oligonucleotide primer for PCR using pAA1 astemplate.

SEQ ID NO:5: Designed oligonucleotide primer for DNA sequencing of pAA1.

SEQ ID NO:6: Designed oligonucleotide primer for amplifying DNA fragment4-3N.

SEQ ID NO:7: Designed oligonucleotide primer for amplifying DNA fragment4-3N.

SEQ ID NO:8: Designed oligonucleotide primer for cloning DNA fragment4-3UN.

SEQ ID NO:9: Designed oligonucleotide primer for cloning DNA fragment4-3UN.

SEQ ID NO:10: Designed oligonucleotide primer for cloning DNA fragment4-3C.

SEQ ID NO:11: Designed oligonucleotide primer for cloning DNA fragment4-3C.

SEQ ID NO:12: Nucleotide sequence of ORF in agarase 1-7 gene.

SEQ ID NO:13: Nucleotide sequence of ORF in agarase 4-3 gene.

SEQ ID NO:14: Amino acid sequence of agarase 1-7.

SEQ ID NO:15: Amino acid sequence of agarase 4-3.

SEQ ID NO:16: Designed oligonucleotide primer for amplifying DNAfragment from 16S ribosomal.

SEQ ID NO:17: Designed oligonucleotide primer for amplifying DNAfragment from 16S ribosomal.

SEQ ID NO:18: Designed oligonucleotide for constructing plasmid forexpressing agarase 1-7.

SEQ ID NO:19: Designed oligonucleotide for constructing plasmid forexpressing agarase 1-7.

SEQ ID NO:20: Designed oligonucleotide for constructing plasmid forexpressing agarase 1-7.

SEQ ID NO:21: Designed oligonucleotide for constructing plasmid forexpressing agarase 4-3.

SEQ ID NO:22: Designed oligonucleotide for constructing plasmid forexpressing agarase 4-3.

SEQ ID NO:23: Designed oligonucleotide for constructing plasmid forexpressing agarase 4-3.

SEQ ID NO:24: Designed oligonucleotide for constructing plasmid forexpressing agarase 1-7.

SEQ ID NO:25: Designed oligonucleotide for constructing plasmid forexpressing agarase 1-7.

SEQ ID NO:26: Designed oligonucleotide for constructing plasmid forexpressing agarase 1-7.

SEQ ID NO:27: Designed oligonucleotide for constructing plasmid forexpressing agarase 1-7.

SEQ ID NO:28: Designed oligonucleotide for constructing plasmid forexpressing agarase 4-3.

SEQ ID NO:29: Designed oligonucleotide for constructing plasmid forexpressing agarase 4-3.

SEQ ID NO:30: Designed oligonucleotide for constructing plasmid forexpressing agarase 4-3.

1-6. (canceled)
 7. A gene encoding an α-agarase, selected from the groupconsisting of: (1) a gene encoding a polypeptide containing an aminoacid sequence consisting of 749 residues from residue 177 to residue 925in the amino acid sequence of SEQ ID NO:14; (2) a gene containing anucleotide sequence consisting of 2247 bases from nucleotide 529 tonucleotide 2775 in the nucleotide sequence of SEQ ID NO:12; and (3) agene that hybridizes to the gene of (2) in a solution containing 6×SSC(1×SSC:0.15M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhardt's and 100 mg/ml herring sperm DNA at 65° C. 8-10. (canceled)11. A recombinant DNA molecule which contains the gene defined by claim7.
 12. A transformant harboring the recombinant DNA molecule defined byclaim
 11. 13. A method for producing α-agarase comprising culturing atransformant defined by claim 12 and collecting the α-agarase from theculture. 14-19. (canceled)